Typhoidal and non-typhoidal salmonellosis continues to cause significant morbidity and mortality worldwide. Our preliminary data establish that the alternative sigma factor sigma E plays a critical role in defending Salmonella from products of the phagocyte respiratory burst, a critical component of host defense against Salmonella. At least 16 genes are positively- or negatively-regulated by sigma E, but most of these are presently unidentified. The specific aims of this proposal are to: (1) Identify sigma E-regulated genes- Both genetic and protein- based methods will be used to identify novel sigma E-regulated loci, which will be mutated by allelic replacement. (2) Genetically analyze the sigma E regulatory network- Studies will be performed to identify signals activating sigma E in vivo, clarify interactions with stationary phase regulatory networks, and identify intermediary loci responsible for negative gene regulation by sigma E. (3) Analyze the roles of sigma E and sigma E-regulated genes in Salmonella pathogenesis- Resistance to oxidant stress conditions, survival in phagocytes, and virulence in mice will be evaluated. (4) Assess the immunogenicity of sigma E-deficient S. typhimurium- The efficacy of rpoE mutant vaccine constructs will be assessed, and preliminary studies to determine the feasibility of a Salmonella-based E. coli O157:H7 vaccine will be performed. The overall goal of this project is to understand the mechanisms by which the sigma E regulon mediates bacterial resistance to oxygen-dependent phagocyte killing. Studies of interactions between Salmonella and phagocytic cells will help to identify novel strategies for the therapy and prevention of salmonellosis. The sigma E regulon is highly conserved among Gram-negative bacteria, making these observations in Salmonella applicable to diverse microbial systems.